The ever-increasing world population and the dwindling supply of arable land available for agriculture fuel research towards improving the efficiency of agriculture. Conventional means for crop and horticultural improvements utilise selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are typically labour intensive and result in plants that often contain heterogeneous genetic components that may not always result in the desirable trait being passed on from parent plants. Advances in molecular biology have allowed mankind to modify the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant. Such technology has the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits. A trait of particular economic interest is yield. Yield is normally defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality. Yield is directly dependent on several factors, for example, the number and size of the organs, plant architecture (for example, the number of branches), seed production and more. Root development, nutrient uptake and stress tolerance are also important factors in determining yield. Crop yield may be increased by optimizing one of the abovementioned factors, which may be done by modifying the inherent growth mechanisms of a plant.
The inherent growth mechanisms of a plant reside in a highly ordered sequence of events collectively known as the ‘cell cycle’. Progression through the cell cycle is fundamental to the growth and development of all multicellular organisms and is crucial to cell proliferation. The major components of the cell cycle are highly conserved in yeast, mammals, and plants. The cell cycle is typically divided into the following sequential phases: G0-G1-S-G2-M. DNA replication or synthesis generally takes place during the S phase (“S” is for DNA synthesis) and mitotic segregation of the chromosomes occurs during the M phase (the “M” is for mitosis), with intervening gap phases, G1 (during which cells grow before DNA replication) and G2 (a period after DNA replication during which the cell prepares for division). Cell division is completed after cytokinesis, the last step of the M phase. Cells that have exited the cell cycle and that have become quiescent are said to be in the G0 phase. Cells in this phase can be stimulated to renter the cell cycle at the G1 phase. The “G” in G1, G2 and G0 stands for “gap”. Completion of the cell cycle process allows each daughter cell during cell division to receive a full copy of the parental genome.
Cell division is controlled by two principal cell cycle events, namely initiation of DNA synthesis and initiation of mitosis. Each transition to each of these key events is controlled by a checkpoint represented by specific protein complexes (involved in DNA replication and division). The expression of genes necessary for DNA synthesis at the G1/S boundary is regulated by the E2F family of transcription factors in mammals and plant cells (La Thangue, 1994; Muller et al., 2001; De Veylder et al., 2002). Entry into the cell cycle is regulated/triggered by an E2F/Rb complex that integrates signals and allows activation of transcription of cell cycle genes. The transition between the different phases of the cell cycle, and therefore progression through the cell cycle, is driven by the formation and activation of different heterodimeric serine/threonine protein kinases, generally referred to as cyclin-dependent kinases (CDK). A prerequisite for activity of these kinases is the physical association with a specific cyclin, the timing of activation being largely dependent upon cyclin expression. Cyclin binding induces conformational changes in the N-terminal lobe of the associating CDK and contributes to the localisation and substrate specificity of the complex. Monomeric CDKs are activated when they are associated with cyclins and thus have a kinase activity. Cyclin protein levels fluctuate in the cell cycle and therefore represent a major factor in determining timing of CDK activation. The periodic activation of these complexes containing cyclins and CDK during cell cycle mediates the temporal regulation of cell-cycle transitions (checkpoints).
Cyclins can be grouped into mitotic cyclins (designated A- and B-type cyclins in higher eukaryotes and CLBs in budding yeast) and G1-specific cyclins (designated D-type cyclins in mammals and CLNs in budding yeast). H-type cyclins regulate the activity of the CAKs (CDK-activating kinases). All four types of cyclins known in plants were identified mostly by analogy to their human counterparts. In Arabidopsis, ten A-type, nine B-type, ten D-type and one H-type cyclin have been described (Vandepoele et al., 2002).
The 10 D-type cyclins are subdivided into seven subclasses, D1 to D7, which reflect their lack of high sequence similarity to each other, which is in contrast to the A-type and B-type cyclins.
Only the D3 and D4 subclasses have different members, respectively three and two. Redundancy of the D3-type cyclins has been proposed previously as an explanation for the failure to observe mutant phenotypes upon knocking out of a single D3-type cyclin (Swaminathan et al., 2000). The two D3-type cyclins are linked via a recent segmental duplication, which suggests that these are functionally redundant. A similar hypothesis could hold for D4-type cyclins, because two out of three are located in a duplicated block.
The much larger divergence seen for D-type cyclins compared with A- and B-type cyclins might reflect the presumed role of D-type cyclins in integrating developmental signals and environmental cues into the cell cycle. For example, D3-type cyclins have been shown to respond to plant hormones, such as cytokinins and brassinosteroids, whereas CYCD2 and CYCD4 are activated earlier in G1 and react to sugar availability (for review, see Stals and Inzé, 2001).
Overexpression of the CYCD2; 1 gene in tobacco was reported to increase cell division and increase overall plant growth rate with no morphological alterations (Cockcroft et al., 2000).
Overexpression in Arabidopsis of the CYCD3; 1 gene under the control of a CaMV 35S promoter was reported to give plants with enlarged cotyledons, a dramatically reduced final plant size and distorted development. At a cellular level, cells are pushed from G1, causing ectopic cell divisions in both meristematic regions and in regions in which cell division normally is absent or limited. This increase of cell numbers is coupled to a decrease in cell size (Dewitte et al., 2003).
The ability to more accurately influence the cell cycle of a plant, and to thereby more accurately modify various growth characteristics of a plant, would have many applications in areas such as crop enhancement, plant breeding, in the production of ornamental plants, aboriculture, horticulture, forestry, the production of algae for use in bioreactors (for the biotechnological production of substances such as pharmaceuticals, antibodies, or vaccines, or for the bioconversion of organic waste) and other such areas.
It is an object of the present invention to overcome some of the problems associated with the prior art expression of cyclin D3 in plants.